Experimental and clinical evidence indicates a critical role for CD4+ T cells (TCD4+) in responses to viruses, in the induction of most effective anti-tumor responses, and in most autoimmune responses. TCD4+ recognize antigen in the form of short peptides (epitopes) held at the surface of the antigen-bearing cell by class II molecules encoded within the major histocompatibility complex and respond with a release of factors that drive and guide the immune response. Antigens can enter the class II antigen presentation pathway from the extracellular milieu and be catabolized in the endosome, possibly the lysosome. Less well understood but clearly operational is a pathway that permits endogenous (intracellular) sources of antigen to be presented in the context of class II. This pathway may provide the basis for novel vaccine design, more effect anti-tumor immunotherapy regimes and improved therapy for autoimmunity. Previously, we have observed distinct antigen processing requirements for three different TCD4+ epitopes of A/PR/834 influenza virus (PR8) presented by the I-Ed class II molecule. The "site 1" epitope of the hemagglutinin (HA) molecule appears to be presented primarily, if not exclusively, from exogenous sources, while the site 1 epitope on the neuraminidase (NA) molecular appears to be presented only from endogenous sources. A second epitope on the HA, site 3, appears to be presented from both sources of antigen. These findings suggest that the presentation pathway(s) an antigen follows is dictated by the susceptibility to proteolysis of both the epitope itself and the full-length protein from which the epitope is derived and the location of the antigen within the cell. To test this hypothesis we will: 1) determine the subcellular locations that exogenous and biosynthesized forms of HA and NA have access to by employing high- definition immunofluorescent confocal microscopy. 2) identify the subcellular compartments in which antigenic peptide is generated from exogenous and endogenous forms of the antigens by employing recently developed biochemical techniques, 3) move these epitopes into new protein contexts to determine the extent to which the epitopes themselves dictate the presentation pathways that are utilized, and 4) direct HA and NA to different regions of the cell, employing site-directed mutagenesis, to elucidate the impact that subcellular location has upon the ability of an antigen to access the endogenous presentation pathway. Our study of three well-defined epitopes, all restricted to the same class II element, our plan to attack the main hypothesis from several different angles, and our use of a system that permits in vivo experiment, put us in a strong and unique position to obtain relevant information about he class II-restricted endogenous presentation pathway. We are particularly interested in understanding what features of a protein determine its ability to be presented through an endogenous route and how this pathway might be manipulated to maximize, quantitatively and qualitatively, the display of internal antigens. This information in turn could be used to advantage across a wide spectrum of health areas.